The metabolism of ethanol perturbs the major nucleotides of liver, the pyridine nucleotides (NAD, NADH) and the adenine nucleotides (ATP, ADP, AMP). Moreover, the distributions of adenine nucleotides are central to considerations of cellular bioenergetics, and accurate quantitative data for the subcellular distributions of enzymes is essential in the analysis of metabolism in vivo. Compared with traditional techniques of tissue homogenization, digitonin fractionation of isolated hepatocytes provides a much more rapid and, in some instances, more accurate determination of enzyme compartmentation. Results with ATP citrate lyase illustrate the information that uniquely can be obtained. Although previously thought to be entirely cytosolic, digitonin fractionation has shown that a portion of total cellular ATP citrate lyase is bound to mitochondria or some other structure, and the amount bound varies with the animal's nutritional state. In hepatocytes from rats that were either starved two days, fed NIH stock diet ad libitum, or starved two days then fed a fat free diet two days, the noncytosolic activity was, respectively, 52, 21, or 24 percent of total cellular lyase. However, because starvation/refeeding greatly induces lipogenic enzymes, the amount of bound lyase activity in this dietary state was 10-12 times greater than that in rats that were starved or fed ad libitum. The association of citrate lyase with a subcellular organelle is also influenced by CoA. Addition of 20 MuM CoA to the digitonin fractionation medium caused all of the lyase to be released from cells like a cytosolic enzyme. Conversely, when cellular free CoA was decreased by incubating hepatocytes with the hypolipidemic agent, 5-(tetradecyloxy)-2-furoic acid, the amount of bound lyase was increased. These results suggest the possibility that the noncytosolic ATP citrate lyase may have a special role in lipogenesis. It has been found that intracellular compartmentaion and metabolic zonation can effectively be studied by perfusing the intact liver with digitonin.